Plant biomass is the most abundant source of carbohydrate in the world due to the lignocellulosic materials comprising the cell walls of all higher plants. Plant cell walls are divided into primary and secondary cell walls. The primary cell wall, which provides structure for expanding cells (and hence changes as the cell grows), is composed of three major polysaccharides, cellulose, hemicellulose and pectin, and one group of glycoproteins. The secondary cell wall, which is produced after the cell has completed growing, also contains polysaccharides and is strengthened through polymeric lignin covalently cross-linked to hemicellulose.
Hemicellulose is a general term used to refer to cell wall polysaccharides that are not celluloses or pectins. Hemicellulose sugar backbones include a variety of compounds, including xylans, xyloglucans, arabinoxylans and mannans. One of the chief constituents of hemicellulose is the aldohexose glucose, mannose, which also may be in the form of a pyranose ring structure, β-D-mannose. With respect to mannose, the glycosidic linkage is on the 1-carbon as a β-bond, having available linkage sites at the 2-, 3-, 4-, and 6-carbons.
A particularly rich source of mannans is the hemicellulose content of softwood, and in particular, the waste material from softwood processing in paper manufacturing. One of the more important hemicelluloses in softwood is galactoglucomannan, which is composed of a backbone of β-(1,4)-linked D-mannopyranose and D-glucopyranose in a ratio of approximately 3:1, respectively (Sjostrom, E. (1992) Wood Chemistry, 2nd Ed., Academic Press: New York, N.Y., pp 63–70). Other sources of mannans include the endosperm of copra and ivory palm nuts, guar beans, coffee beans, and roots of konjak (Amorphorphallus konjac).
Enzymatic degradation of the β-linkages in β-1,4-D-mannans requires the coordinate action of several mannanases. Mannanases have been identified in Bacillus (Emi et al., (1972) Agr. Biol. Chem. 36:991–1001), Aeromonas (Araki et al., (1983) Agr. Kyushu Univ. 27:89–98), Streptomyces (Takahashi et al., (1984) Biol. Chem. 48:2189–2195) and several fungal species (Hashimoto et al., (1969) J. Nippon Nogeikagaku Kaishi 43:317–322).
Mannanases are given an Enzyme Commission (EC) designation according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (Eur. J. Biochem. 264:607–609 and 610–650 (1999)). β-mannosidase (EC 3.2.1.25) (Hylin et al., (1964) J. Biol. Chem., 239:990) and β-mannanase (EC 3.2.1.78) (Reese, E. T., (1965) Can. J. Microbiol. 11:167) cleave the β-mannoside linkages in β-1,4-D-mannans to yield D-mannose and manno-oligosaccharides, respectively. Other mannanase activities have been identified, for example, exomannanase (1,4-β-D-Mannan mannohydrolase) (EC 3.2.1.xx—unassigned) and exomannobiohydrolase (1,4-β-D-Mannan mannobiohydrolase) (EC 3.2.1.100) (McCleary, B. V., (1988) Methods Enzymol. 160:589–595; Araki et al., (1982) J. Biochem. 91:1181).
Industrial applications of hemicellulases, and mannanases in particular, are primarily targeted at situations where selective removal of hemicellulose is required to elevate the value of a complex substrate, such as in foods, feeds, and paper pulp. Food industry applications include the processing of coffee (Godfrey, T, (1983) in Industrial Enxymology: The Applications of Enzymes in Industry, Godfrey and Reichelt, eds., MacMillan Press: Basingstoke, UK, pp 340–351) the maceration of fruits and vegetables (Biely, P, (1985) Trends Biotechnol. 3:286–290), and bread preparation (Maat et al., (1992) Xylans and Xylanases, Visser, J., Beldman M., Kusters-van Someren and Voragen, eds., Elsevier: New York, N.Y., pp 349–360). Feed industry applications includes the processing of poultry feed (van Paridon et al., (1992) Xylans and Xylanases, Visser, J., Beldman M., Kusters-van Someren and Voragen, eds., Elsevier: New York, N.Y., pp 371–378).
In addition, mannanases can be useful in the production of biofuels from plant biomass, where the mannanases participate in the hydrolysis of the hemicellulose fraction to simpler sugars, which are then converted to ethanol via fermentation.
Highly thermostable enzymes have been isolated from the thermophile Acidothermus cellulolyticus gen. nov., sp. nov., a bacterium originally isolated from decaying wood in an acidic, thermal pool at Yellowstone National Park (Mohagheghi et al., (1986) Int. J. Systematic Bacteriology 36(3):435–443). One cellulase enzyme produced by this organism, the endoglucanase EI, is known to display maximal activity at 75° C. to 83° C. (Tucker et al., Bio/Technology, 7(8):817–820). E1 endoglucanase has been described in U.S. Pat. No. 5,275,944. The A. cellulolyticus E1 endoglucanase is an active cellulase; in combination with exocellulase CBH I from Trichoderma reesei, E1 gives high levels of saccharification and contributes to a degree of synergism. Baker et al., (1994) Appl. Biochem. Biotechnol. 45/46:245–256. The gene encoding E1 catalytic and cellulose binding domains and linker peptide were described in U.S. Pat. No. 5,536,655. The potential exists for the successful, commercial scale expression of heterologous mannanases, and in particular thermal stable mannanases. Like the E1 endoglucanase, thermal stable mannanases would likely have the desirable characteristic of maximal activity at elevated temperatures, as well as potentially having the thermal tolerant associated properties of resistance to acid inactivation, proteolytic inactivation, and solvent inactivation (Cowan D A in Danson M J et al. (1992) The Archaebacteria, Biochemistry and Biotechnology at 149–159, University Press, Cambridge, ISBN 1855780100). E1 has also been expressed as a stable, active enzyme from a wide variety of hosts, including E. coli, Streptomyces lividans, Pichia pastoris, cotton, tobacco, and Arabidopsis (Dai Z, Hooker B S, Anderson D B, Thomas S R. Transgenic Res. 2000 February; 9(1):43–54).
There is a need within the art to generate alternative mannanase enzymes capable of assisting in the commercial scale processing of mannans to simpler sugars for use in the food, feed, paper pulp and biofuels industries. Against this backdrop the present invention has been developed.